Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Immunocytochemistry and quantification of protein colocalization in cultured neurons

Abstract

This protocol details a method to quantify the distribution of protein and colocalization in neuronal cultures. To that end, this protocol includes itemized steps and considerations for performing immunocytochemistry, acquiring fluorescence images and quantifying multichannel fluorescence images. Success in quantifying immunostained neurons relies on the accessibility of the proteins of interest, the sensitivity and specificity of antibodies, the signal-to-noise ratio of the collected image and the sensitivity of the quantification method. In contrast to other commonly employed methods for quantification, the protocol detailed here requires manual selection of punctae and subtraction of background selected for each neurite. This approach reliably and uniquely allows for detection of proteins in low signal-to-noise ratio images, which are characteristic of developing neurons. Thus, this method serves an important niche in image analysis poorly addressed by alternative published methods. In general, immunocytochemistry requires 3.5–7 h, and one triple-immunostained neuron can be quantified in 1.5 h.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Flow chart for the various immunostaining methods to detect surface or total protein.
Figure 2: Controlling for cell membrane permeabilization is a critical step in characterizing surface protein expression.
Figure 3: Different methods of background selection can markedly alter measures of protein density and colocalization.
Figure 4: Selection of background ROIs and construction of a multichannel punctae mask.

Similar content being viewed by others

References

  1. Rao, A., Kim, E., Sheng, M. & Craig, A.M. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J. Neurosci. 18, 1217–1229 (1998).

    Article  CAS  Google Scholar 

  2. Liao, D., Zhang, X., O'Brien, R., Ehlers, M.D. & Huganir, R.L. Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat. Neurosci. 2, 37–43 (1999).

    Article  CAS  Google Scholar 

  3. Washbourne, P., Bennett, J.E. & McAllister, A.K. Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat. Neurosci. 5, 751–759 (2002).

    Article  CAS  Google Scholar 

  4. Washbourne, P., Liu, X.B., Jones, E.G. & McAllister, A.K. Cycling of NMDA receptors during trafficking in neurons before synapse formation. J. Neurosci. 24, 8253–8264 (2004).

    Article  CAS  Google Scholar 

  5. Trimmer, J.S., Trowbridge, I.S. & Vacquier, V.D. Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca2+ and H+ fluxes of sea urchin sperm. Cell 40, 697–703 (1985).

    Article  CAS  Google Scholar 

  6. Elmariah, S.B., Crumling, M.A., Parsons, T.D. & Balice-Gordon, R.J. Postsynaptic TrkB-mediated signaling modulates excitatory and inhibitory neurotransmitter receptor clustering at hippocampal synapses. J. Neurosci. 24, 2380–2393 (2004).

    Article  CAS  Google Scholar 

  7. Banker, G. & Goslin, K. (eds.). Culturing Nerve Cells 2nd edition. (MIT Press, Cambridge, MA, 1998).

    Google Scholar 

  8. Spector, D.L., Goldman, R.D., Leinwand, L.A. (eds.). Cells, A Laboratory Manual: Subcellular Localization of Genes and Their Products Vol. 3. (Cold Spring Harbor Laboratory Press, Plainview, NY, 1998).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A Kimberley McAllister.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Glynn, M., McAllister, A. Immunocytochemistry and quantification of protein colocalization in cultured neurons. Nat Protoc 1, 1287–1296 (2006). https://doi.org/10.1038/nprot.2006.220

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.220

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing